Asparagine-linked protein glycosylation is involved in a wide range of biological processes that are implicated in human health and disease. The modification is essential in all aspects of human development and homeostasis and now recognized to be involved in bacterial pathogenicity and viral infection and survival. N-linked glycosylation is also of great relevance in medicine and biotechnology, as protein therapeutics including monoclonal antibodies, glycoprotein hormones, and cytokines have become important elements in the modern pharmacopoeia. While the specific biological functions and the molecular details of N-linked glycosylation in bacteria and man vary, there are key conserved elements in the overall processes that support the hypothesis that the detailed study of the prokaryotic protein glycosylation (pgl) pathway of C. jejuni, which forms the basis for this research, will also provide insight into the more complex process of N-linked glycosylation in eukaryotes. The biosynthetic pathways that lead to N-linked glycosylation in all organisms involve multistep membrane- associated processes and feature complex highly lipophilic polyprenol-linked substrates. These characteristics pose major challenges as we seek to develop a detailed molecular understanding of how the enzymes in these pathways come together to afford the ordered biosynthetic assembly line that ultimately yields essential N-linked glycoproteins. Until we understand the relationships amongst the polyprenol-linked substrates, the enzymes that process them and the membrane bilayer in which they are localized, we will be limited in our ability to target the constituent processes to mitigate disease and to exploit th pathways to generate glycoproteins that might be relevant as therapeutic biologicals. The proposed research centers on the application of synergistic in vitro and in vivo biochemical and biophysical approaches for investigating bacterial N-linked protein glycosylation. The aims of the proposal are: 1. To investigate the structural and functional interactions of the five pgl pathway glycan assembly enzymes in a Nanodisc model membrane system and to define the functional significance of conserved linear long-chain polyprenyl moieties in the substrates of the pathway enzymes. 2. To apply in vivo fluorescence-based imaging to correlate findings from the interaction analysis in the in vitro model membrane with studies in E. coli where the pgl pathway has been functionally reconstituted. 3. To define the interactions of the OTase, PglB with cognate substrates in vitro in Nanodisc membranes and in vivo with the membrane-associated proteins of the pgl pathway and the bacterial translocon. If the goals of the proposed research are achieved, the research will result in the development of general strategies for dissecting related membrane-associated polyprenol-dependent biosynthetic processes that feature prominently in all living systems.